Testosterone, Free, Bioavailable and Total (LC-MS/MS) Test
The Testosterone, Free, Bioavailable and Total (LC-MS/MS) Test measures all three circulating fractions using the gold-standard LC-MS/MS method. It is ordered to diagnose male hypogonadism, resolve equivocal total-testosterone results when SHBG is altered, assess hyperandrogenism in women, and monitor replacement therapy, with superior specificity and sensitivity to immunoassay.
3
Biomarkers
Blood (serum)
Sample
3–5 business days
Turnaround
No
Fasting
What is the Testosterone Panel (LC-MS/MS) test?
The Testosterone, Free, Bioavailable and Total (LC-MS/MS) Test measures all three fractions of circulating testosterone using liquid chromatography-tandem mass spectrometry, the gold-standard analytical method that eliminates the cross-reactivity and poor sensitivity of older immunoassays. Practitioners order this panel to diagnose and monitor male hypogonadism, to resolve discrepancies when total testosterone is borderline and symptoms do not match, and to assess androgen status accurately in settings where SHBG is altered. In male hypogonadism the diagnosis rests on the combination of symptoms — reduced libido, erectile dysfunction, low energy, loss of muscle mass, mood changes — and consistently low testosterone measured on two separate morning samples. Total testosterone is the initial screen, but when it falls into the equivocal zone (usually 200–400 ng/dL) or when SHBG is known to be high or low, free or bioavailable testosterone becomes decisive. LC-MS/MS methodology ensures that results are reliable even at the low concentrations seen in women, in whom the test may be ordered to investigate hyperandrogenism, and in children, where immunoassays lack the necessary sensitivity.
What does the Testosterone Panel (LC-MS/MS) test measure?
Total testosterone represents the sum of free, albumin-bound, and sex hormone-binding globulin (SHBG)-bound fractions. Approximately 98% circulates bound — roughly 40–50% tightly to SHBG, 50–60% loosely to albumin — leaving 1–2% unbound. Liquid chromatography-tandem mass spectrometry (LC-MS/MS) offers superior specificity and sensitivity compared with immunoassay, particularly at low concentrations in women and children, and avoids cross-reactivity with other steroids.
Total testosterone is the initial marker of androgenisation and the first-line test in the evaluation of male hypogonadism, where it is interpreted with clinical symptoms. Because most testosterone is protein-bound, total testosterone alone may not reflect tissue-available androgen when SHBG is altered by obesity, ageing, liver disease, or thyroid dysfunction, so practitioners often measure free or bioavailable testosterone for a more accurate picture of androgen status.
Free testosterone is the unbound, biologically active fraction that diffuses freely into target tissues to exert genomic and non-genomic androgenic effects. Direct immunoassay methods for free testosterone are unreliable; calculation from total testosterone and SHBG (with albumin assumed) via equilibrium equations is widely accepted, though equilibrium dialysis and ultrafiltration remain reference methods.
Free testosterone is the best marker of bioavailable androgen when SHBG is altered. A man with a borderline-low total testosterone and high SHBG (for example, due to ageing or hyperthyroidism) may have a low-normal free testosterone and few symptoms, whereas obesity-associated low SHBG can produce a low total testosterone with preserved free testosterone. Calculating or directly measuring free testosterone resolves these discrepancies and guides treatment decisions more accurately than total testosterone alone.
Bioavailable testosterone is the sum of free testosterone plus the albumin-bound fraction, both of which dissociate readily and are considered tissue-available. Because albumin binding is of much lower affinity than SHBG binding, albumin-bound testosterone equilibrates quickly with the free pool at target tissues. Bioavailable testosterone can be measured by ammonium sulphate precipitation of SHBG-bound steroid or calculated from total testosterone, SHBG, and albumin.
Bioavailable testosterone offers a practical middle ground between total and free testosterone, capturing the androgen that is immediately accessible to tissues. In conditions that alter SHBG — obesity, metabolic syndrome, liver disease, thyroid dysfunction — bioavailable testosterone often correlates better with clinical signs of androgen deficiency or excess than does total testosterone, and some practitioners prefer it for the assessment of equivocal cases.
Why is the Testosterone Panel (LC-MS/MS) test ordered?
- Evaluation of suspected hypogonadism in men with sexual dysfunction, reduced libido, fatigue, loss of muscle mass, or osteoporosis, where symptoms may not match a borderline total testosterone
- Confirmation of low total testosterone found on initial screening, with a repeat morning sample before any therapy is considered
- Assessment of androgen status in men with obesity, metabolic syndrome, or type 2 diabetes, where low SHBG may depress total testosterone while free or bioavailable testosterone remains near-normal
- Monitoring of testosterone replacement therapy to ensure physiological free and bioavailable concentrations, not just total testosterone normalisation
- Investigation of hyperandrogenism in women presenting with hirsutism, acne, or menstrual irregularity, where LC-MS/MS offers the necessary sensitivity and specificity
- Evaluation of delayed or precocious puberty in paediatric and adolescent patients, where accurate measurement at low concentrations is essential
- Assessment in older men with equivocal symptoms and borderline results, where rising SHBG with age can mask free-testosterone deficiency
- Pre-treatment evaluation and ongoing monitoring in patients on medications that alter SHBG (opioids, glucocorticoids, anticonvulsants) or in chronic illness affecting androgen production
Sample collection and turnaround
Sample type
Blood (serum)
Fasting required
No
Collection method
Venipuncture at patient service centre
Turnaround
3–5 business days
Collection notes
Draw in the morning, ideally before 10:00, to account for diurnal variation when assessing hypogonadism. Document testosterone or anabolic steroid use, opioids, glucocorticoids, and high-dose biotin. Defer testing during acute illness where possible, and confirm a low result on a separate morning sample.
Specimen requirements
Minimum 1.0 mL serum in a red-top or gel separator tube. Separate from cells promptly and refrigerate or freeze if analysis is delayed. LC-MS/MS analysis is robust to most storage conditions, but prolonged room-temperature storage should be avoided.
What can affect Testosterone Panel (LC-MS/MS) results?
Testosterone peaks in the early morning and declines through the day, so afternoon or evening samples can be 20–30% lower; guideline-concordant evaluation of hypogonadism requires an early-morning draw, ideally before 10:00.
Systemic illness, surgery, or critical care transiently suppresses the hypothalamic–pituitary–gonadal axis, lowering testosterone; defer testing until recovery to avoid misdiagnosing transient suppression as primary hypogonadism.
Obesity lowers SHBG through insulin resistance and increases aromatase-mediated conversion of testosterone to oestradiol in adipose tissue, lowering total and sometimes free testosterone; weight loss can partially reverse the effect.
Some immunoassay platforms (though not LC-MS/MS) use biotin–streptavidin chemistry, so high-dose biotin (>5 mg/d) can interfere; LC-MS/MS avoids this, but practitioners should document biotin use if any immunoassay components are run in parallel.
Testosterone replacement or abuse of anabolic steroids raises measured testosterone and suppresses LH and FSH; document all androgen use before interpretation and expect artefactually high results during therapy.
Opioids suppress the gonadotrophin axis centrally, causing secondary hypogonadism; glucocorticoids suppress ACTH and may secondarily affect the gonadal axis and lower SHBG, compounding the interpretation challenge.
What do Testosterone Panel (LC-MS/MS) results mean?
Elevated
Elevated total, free, or bioavailable testosterone in men may reflect exogenous androgen administration (replacement or abuse); the rare androgen-secreting tumour of testis or adrenal; or resistance syndromes. In women, hyperandrogenism — commonly from polycystic ovary syndrome, congenital adrenal hyperplasia, or androgen-secreting tumour — presents with elevated testosterone, often modest, but clinically significant given the much lower female range; further evaluation with DHEA-S, 17-hydroxyprogesterone, and imaging is warranted.
Normal
Testosterone in the mid-normal range with appropriate free and bioavailable fractions is reassuring in the evaluation of suspected hypogonadism, though symptoms should still be addressed through other lenses — mood disorders, sleep apnoea, thyroid dysfunction, anaemia — since some men with low-normal testosterone do experience symptoms. Normal results in a woman presenting with hirsutism suggest a diagnosis other than frank hyperandrogenism, such as idiopathic hirsutism or increased peripheral androgen sensitivity.
Reduced
Low total testosterone with low or low-normal free and bioavailable testosterone, confirmed on a repeat morning sample and accompanied by symptoms, establishes a diagnosis of hypogonadism. Measure LH and FSH to distinguish primary gonadal failure (high gonadotrophins) from secondary hypothalamic–pituitary causes (low or inappropriately normal gonadotrophins). Isolated low total testosterone with preserved free or bioavailable testosterone, especially in obese men with low SHBG, may not require treatment and should be correlated with clinical symptoms.
Note: Total testosterone alone is insufficient in men with altered SHBG; always interpret free or bioavailable testosterone alongside it. A single low result does not establish hypogonadism — repeat testing on a second morning, check LH and FSH, and correlate with clinical symptoms. Avoid testing during acute illness. LC-MS/MS eliminates many assay interferences, but biological variation and diurnal rhythm remain, so consistency in draw time and laboratory is important for serial monitoring.
Frequently asked questions
LC-MS/MS offers superior specificity and sensitivity, particularly at low concentrations in women and children, and eliminates cross-reactivity with other steroids that can falsely elevate immunoassay results. It is the preferred method for accurate testosterone measurement.
How to access the Testosterone Panel (LC-MS/MS) test
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Published: October 7, 2026
Last reviewed: October 7, 2026
- Bhasin S, et al. Testosterone therapy in men with hypogonadism: an Endocrine Society clinical practice guideline. J Clin Endocrinol Metab. (2018)
- Stanworth RD, Jones TH. Testosterone for the aging male; current evidence and recommended practice. Clin Interv Aging. (2008)
- Rosner W, et al. Position statement: utility, limitations, and pitfalls in measuring testosterone — an Endocrine Society position statement. J Clin Endocrinol Metab. (2007)
- Vesper HW, et al. Harmonization of testosterone measurements in humans. J Clin Endocrinol Metab. (2022)
- Mulhall JP, et al. Evaluation and management of testosterone deficiency: AUA guideline. J Urol. (2018)
This content is for licensed healthcare practitioners only and does not constitute medical advice. Clinical decisions should be based on the full clinical picture and not on any single test result.
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